Determination of oligonucleotides for therapeutics, diagnostics and research reagents

ABSTRACT

Oligonucleotides which selectively bind to target biomolecules are determined by in vitro assay of a pool of random oligonucleotides for activity against the biomolecules, followed by recovery and characterization of selected oligonucleotides. Oligonucleotides so determined may be utilized for therapeutic, diagnostic and research reagent purposes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 08/330,000 filed Oct.27, 1994, U.S. Pat. No. 5,686,242, which is a continuation-in-part ofPCT/US92/07489 filed Sep. 4, 1992 which is a continuation-in-part ofU.S. Ser. No. 07/755,485 filed Sep. 5, 1991, now abandoned, all whichare incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

This invention relates to in vitro selection of oligonucleotidesequences which selectively bind to a target biomolecule underphysiological conditions. In accordance with preferred embodiments,therapeutic oligonucleotides are identified.

BACKGROUND OF THE INVENTION

The development of new drugs and biologically active substances fortherapeutic, diagnostic and research reagent purposes traditionallyconcentrates on the rational design of such compositions based uponknowledge of the target biomolecule, i.e. the molecule to be modulated.

Bodily states in mammals, including many disease states, are effected bybiomolecules. Classical therapeutics has generally focused uponinteractions with proteins in efforts to modulate disease-potentiatingfunctions of such proteins. In addition to modulatingdisease-potentiating functions of proteins directly, recent attemptshave been made to moderate the actual production of proteins byinteractions with molecules that direct their synthesis, intracellularRNA. Biological functions may also be modulated or regulated byinteractions with other biomolecules such as nucleic acids,carbohydrates, lipids, steroids or toxins.

One approach for constructing therapeutics, diagnostics and researchreagents has been simple modifications of known amino acid or nucleicacid sequences. Such techniques are limiting because the number ofindividual sequences of simple modifications necessary for thedevelopment of new substances is prohibitively large. In addition, manydrug targets and other target molecules are too extensive and complex tobe analyzed by these mutational experiments. Other biomolecule targets,by virtue of their particular chemical nature, are not candidates fordirected mutagenesis of this sort. Some examples of such biomoleculesare carbohydrates, lipids, and steroids.

Recently, methods have been devised whereby therapeutics, diagnosticsand research reagents can be developed more quickly. A variety ofcombinatorial strategies have been described to identify activepeptides. Houghton, et al. Nature 1991, 354, 84; Lam, et al., Nature1991, 354, 82; Owens, et al., Biochem. Biophys. Res. Commun. 1991, 181,402; Fodor, et al., Science 1991, 251, 767; Geysen, et al., MolecularImmunology 1986, 23, 709; Zuckermann, et al., Proc. Natl. Acad. Sci.1992, 89, 4505; Rutter, et al., U.S. Pat. No. 5,010,175 issued Apr. 23,1991; Lam, et al., PCT US91/06444 filed Jul. 1, 1991, Dooley, et al.,Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 10811; Dooley and Houghten,Life Sciences, 1993, 52, 1509; Ohlmeyer, et al., Proc. Natl. Acad. Sci.U.S.A., 1993, 90, 10922; Jayawickreme, et al., Proc. Natl. Acad. Sci.U.S.A., 1994, 91, 1614.

Rutter, et al. describes a method whereby statistically randomizedpeptides may be prepared and active peptide selected for and identified.

Lam, et al., teaches the preparation of randomer libraries, especiallyrandom peptide libraries in which each randomer sequence is individuallycoupled to a solid support (i.e. one oligomer sequence/one bead).Further, a reporter group is attached to each oligomer/bead in order toidentify active oligomers from inactive oligomers. The randomerlibraries of Lam, et al., PCT US91/06444 filed Jul. 1, 1991, arecontacted with a target biomolecule and active randomers, identified viareporter groups, are isolated using the solid support to manually removethe active randomer from the rest of the library. Lam, et al. furtherteaches that the selected randomer can be characterized such as by Edmandegradation or FAB-MS.

Combinatorial strategies for nucleic acids have also been developed.Such methods generally select for a specific nucleic acid sequence froma pool of random nucleic acid sequences based on the ability of theselected sequence to bind to a target protein. The selected sequencesare then commonly amplified and the selection process repeated until afew strongly binding sequences are identified. Commonly, the pool ofrandom nucleic acid sequences is comprised of short random sequencesembedded in external flanking or "carrier" nucleic acid molecules ofknown sequence. Such carrier molecules are intended to neither enhance,nor detract from binding of the oligonucleotide. The amplificationprimers are prepared to be complementary to known sequences of the"carrier" generally of lengths 20 nucleotides in length or more. Such"carrier" portions are meant to facilitate manipulation of the moleculeand preferably have neutral effect upon the randomized sequence to beselected for. Using this method Tuerk and Gold, Science 1990, 249,505;identified a sequence which strongly binds T4 polymerase bindingprotein, gp43, but which would not have been predictable usingtraditional methods. Ellington and Szostak, Nature 1990, 346, 818;identified sequences which bind small ligands using this method, Bock,et al., Nature 1992, 355, 564; designed DNA molecules which recognizethe protease thrombin and Schneider, et al., J. Mol. Biol., 1992, 228,862; isolated RNA ligands with high affinity for the bacteriophage R17coat protein. More recently, using this method, Schneider, et al., FASEBJ., 1993, 7, 201; identified RNA molecules from a pool of RNA moleculesthat bind tightly to the E. coli transcription termination factor rhoand Jellinek, et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 11227;isolated RNA ligands with low-nanomolar affinity and high specificity tobasic fibroblast growth factor from a library of RNA molecules.

Randomer oligonucleotide libraries are also mentioned by Lam, et al.Characterization of selected oligonucleotides after manual separation issuggested using the techniques of Maxam and Gilbert or by the use of anoligonucleotide sequencer. Electrospray-high performance massspectrometry is also suggested in order to determine sequences andstructures of randomers. These methods require the addition of areporter group to the oligomer species in order to identify and manuallyisolate active oligomers. Furthermore, randomer libraries are limited tooligomers approximately 5 mers in length since preparation of more than5 mers would present unwieldy amounts of oligomer/beads as well as theneed for large amounts of target.

While some advantages have been achieved by the foregoing methods,simple methods of determining therapeutics, diagnostics, and researchreagents are desirable. Previous methods have relied upon complexnucleic acid molecules comprised of at least partially random sequenceportions to be selected for, as well as lengthy flanking carrierportions necessary to support the reactions. Still other methods arelimited in the length of oligonucleotides within the randomer librarywhich is physically possible. Methods requiring enzymatic amplificationof active sequences are often limited by incompatibility with manynon-standard nucleotides or nucleosides.

Methods in which selection may be carried out with a fullysequence-randomized pool of discrete molecules and a minimum of otherfixed sequences would be greatly desirable as they would increasespecificity, facilitate manipulation of the pool of molecules, andeliminate any indeterminate bias in binding selection resulting from thepresence of either internal sequence fixed (unrandomized) positionsand/or the carrier portions.

Such improved methods to determine oligonucleotides for antisensetherapeutics, diagnostics and research reagents are greatly desired.

Although oligonucleotides are currently being administered astherapeutic agents, it is not known a priori how to select an idealnucleotide sequence to bind selectively to a target molecule. Moreover,in many cases, it is even difficult to decide what region of a gene orprotein, for example, to target in order to achieve maximum effect. Themethods of the present invention overcome these difficulties.

SUMMARY OF THE INVENTION

In accordance with some embodiments of the present invention, methods ofdetermining oligonucleotides that target the sequence and/or structureof a target biomolecule and have specific activity for that targetbiomolecule are provided. In accordance with said methods a set ofrandomized oligonucleotides may be prepared and assayed for activityagainst the target biomolecule. Active and inactive oligonucleotides areseparated by microanalytical techniques and the active oligonucleotidesare characterized by microanalytical structure determination. In otherembodiments of the present invention, oligonucleotides having specificactivity for a target biomolecule are determined by preparing a set ofrandomized oligonucleotides and assaying the set of randomoligonucleotides for activity against a target biomolecule. Active andinactive oligonucleotides are separated and active oligonucleotides arerecovered. Thereafter active oligonucleotides are amplified by extendinga polyA sequence in a 3' direction on the active oligonucleotide. Afirst chimeric primer comprising a 5' known sequence and a 3' polydTportion may be hybridized to the oligonucleotide and a cDNA strand maybe prepared which is complementary to the oligonucleotide using thefirst chimeric primer. The cDNA may be extended in a 3' direction by theaddition of polyA. Thereafter, the oligonucleotide and cDNA strands areseparated and a second chimeric primer having a 5' known sequence and a3' polydT portion is hybridized to the polyA portion of the cDNAresulting in the formation of recessed 3' ends on both strands. Therecessed ends may be filled to form two complementary strands and thestrands separated and amplified using polymerase chain reaction. Thefirst and second chimeric primers may serve as PCR primers in someembodiments of the present invention. Furthermore, the oligonucleotidestarting product, having been amplified, may be excised from the totalPCR product and may be recovered and used in subsequent assay andamplification steps in order to optimize determination ofoligonucleotides specifically active for a given target biomolecule.

In accordance with other methods of the present invention methods ofdetermining oligonucleotides having specific activity for a targetbiomolecule are provided comprising the steps of preparing a set ofrandomized oligonucleotides and assaying the set of randomizedoligonucleotides for activity against a target biomolecule. Activeoligonucleotides are separated from inactive oligonucleotides andrecovered. Recovered oligonucleotides are characterized to provide anoligonucleotide cassette. Thereafter a set of oligonucleotidescomprising an oligonucleotide cassette and at least one flanking regionof randomized positions are prepared and assayed for activity against atarget biomolecule. Active oligonucleotide are separated from inactiveoligonucleotides and active oligonucleotides are recovered. Thereafterthe recovered oligonucleotides are characterized to provide a newoligonucleotide cassette. This new oligonucleotide cassette may be usedin subsequent preparation, assay and characterization steps in order tooptimize determination of oligonucleotides specifically active for agiven target biomolecule.

In yet further embodiments of the present invention sets of sequenceposition randomized oligonucleotides may be subfractionated and assayedfor activity for a target biomolecule. Oligonucleotides from thesubfraction having the highest activity for a biomolecule may then beassayed, separated from inactive oligonucleotides and characterized inaccordance with methods of the invention.

In other embodiments of the present invention methods of amplifyingoligonucleotides are provided. PolyA may be extended in a 3' directionon the oligonucleotide to be amplified. A first chimeric primercomprising a 5' known sequence and a 3' polydT portion may be hybridizedto the oligonucleotide and a cDNA strand may be prepared which iscomplementary to the oligonucleotide using the first chimeric primer asa primer. The CDNA may be extended in a 3' direction by the addition ofpolyA. Thereafter, the oligonucleotide and cDNA strands are separatedand a second chimeric primer having a 5' known sequence and a 3' polydTportion is hybridized to the polyA portion of the cDNA resulting in theformation of recessed 3' ends on both strands. The recessed ends may befilled to form two complementary strands and the strands separated andamplified using polymerase chain reaction. The first and second chimericprimers may serve as PCR primers in some embodiments of the presentinvention.

In accordance with other embodiments of the present invention, a set ofrandomized oligonucleotides are prepared and allowed to bind to thetarget nucleic acid. The active oligonucleotides are identified andsequenced by mapping the target site to which the oligonucleotides havebound, and this is accomplished by affinity cleavage using enzymatic orchemical cleavage.

In yet other embodiments, affinity mapping may be followed by afootprinting assay aimed at optimizing the sequence and length ofoligonucleotides for enhanced binding to the preferred target site.

In accordance with still other embodiments of the present invention,oligonucleotides identified in accordance with methods of the presentinvention may be employed further in diagnostic and research methods forthe detection of chemicals or drugs present in a sample.

Oligonucleotides identified in accordance with the methods of thepresent invention are also encompassed by the present invention.Oligonucleotides identified and sequenced in accordance with methods ofthe present invention are likewise encompassed by the present invention.In accordance with other embodiments of the present invention diagnosticand research reagents are provided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Methods of determining oligonucleotides that are specifically activeagainst target biomolecules are provided. In the context of the presentinvention "determine" refers to the identification of the sequence ofoligonucleotides and the binding activity of the oligonucleotides for atarget molecule. Further, "determine" refers to the identification ofoligonucleotides having activity such as catalytic or enzymaticactivity. In some instances, neither the oligonucleotide sequence norits specific activity is known prior to performance of methods of thepresent invention. In other cases, while a particular oligonucleotidesequence may be known, those skilled in the art may not recognize itsactivity for a particular target molecule. In still other cases,activity of a known sequence for a particular target molecule may beoptimized.

In some embodiments of the present invention, "specific activity" refersto binding affinity of said oligonucleotides for a target molecule, andfurther encompasses activity such as catalytic or enzymatic activity. Asused herein, binding affinity refers to the ability of the oligomer tobind to a target molecule via hydrogen bonds, van der Waalsinteractions, hydrophobic interactions, or otherwise. For example, anoligonucleotide may have binding affinity for another oligonucleotide towhich it is complementary, i.e., to which it has the ability tohybridize due to Watson-Crick base pair attraction. Thus activeoligonucleotides have specific activity, be it binding, catalytic,enzymatic, or other activity for a target molecule, while inactiveoligonucleotides exhibit minimal or no specific activity for a targetbiomolecule. The amount of activity sufficient to differentiate anactive oligonucleotide from an inactive oligonucleotide will, of course,vary depending upon the parameters of the assay and the biomoleculetargeted.

In accordance with said methods randomized oligonucleotides areprepared. In the context of this invention, the term "oligonucleotide"refers to a polynucleotide formed from naturally occurring bases andfuranosyl groups joined by native phosphodiester bonds. This termeffectively refers to naturally occurring species or synthetic speciesformed from naturally occurring subunits or their close homologs. Theterm "oligonucleotide" may also refer to moieties which have portionssimilar to naturally occurring oligonucleotides but which havenon-naturally occurring portions. Thus, oligonucleotides may havealtered sugar moieties or inter-sugar linkages. Exemplary among theseare the phosphorothioate and other sulfur-containing species which areknown for use in the art. In accordance with some preferred embodiments,at least some of the phosphodiester bonds of the oligonucleotide havebeen substituted with a structure which functions to enhance thestability of the oligonucleotide or the ability of the oligonucleotideto penetrate into the region of cells where the viral RNA is located. Itis preferred that such substitutions comprise phosphorothioate bonds,phosphotriesters, methyl phosphonate bonds, short chain alkyl orcycloalkyl structures or short chain heteroatomic or heterocyclicstructures. Most preferred are CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂,CH₂ --O--N(CH₃)--CH₂, CH₂ --N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂structures. Also preferred are morpholino structures. Summerton, J. E.and Weller, D. D., U.S. Pat. No. 5,034,506 issued Jul. 23, 1991. Inother preferred embodiments, such as the protein-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to aza nitrogen atoms within the polyamide backbone. P. E.Nielsen, et al., Science 1991 254 1497. In accordance with otherpreferred embodiments, the phosphodiester bonds are substituted withother structures which are, at once, substantially non-ionic andnon-chiral, or with structures which are chiral and enantiomericallyspecific. Persons of ordinary skill in the art will be able to selectother linkages for use in practice of the invention.

Pligonucleotides may also include species which include at least somemodified base forms. Thus, purines and pyrimidines other than thosenormally found in nature may be so employed. Similarly, modifications onthe furanosyl portion of the nucleotide subunits may also be effected,as long as the essential tenets of this invention are adhered to.Examples of such modifications are 2'-O-alkyl and 2'-halogen-substitutednucleotides. Some specific examples of modifications at the 2' positionof sugar moieties which are useful in the present invention are OH, SH,SCH₃, F, OCN, O(CH₂)_(n) NH₂, O(CH₂)_(n) CH₃ where n is from 1 to about10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl;Cl, Br, CN, CF₃, OCF₃, O--, S--, or N-alkyl; O--, S--, or N-alkenyl;SOCH₃, SO₂ CH₃ ; ONO₂ ; NO₂ ; N_(3;) NH_(2;) heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Sugar mimetics such ascyclobutyls may also be used in place of the pentofuranosyl group.Nucleotides may also comprise other modifications consistent with thespirit of this invention. Such nucleotides are best described as beingfunctionally interchangeable with, yet structurally distinct from,natural nucleotides. All such nucleotides are comprehended by thisinvention so long as they effectively function as subunits in theoligonucleotide.

In some embodiments of the present invention oligonucleotide groups maybe detectably labeled. Reporter groups can be incorporated aspolymerizable monomer units such as phosphoramidites, usually at the 3'or 5' ends of the oligonucleotides. Labelling may also be performedfollowing completion of combinatorial library synthesis such as bychemical modification such as post-synthesis conjugation. Radiolabelssuch as ³ H, ¹⁴ C, ³⁵ S, ³² P, ³³ P and ¹⁸ O may be incorporated bymethods known to those skilled in the art. For example, oligonucleotidesmay be labeled at their 5' termini using [γ-³² P] ATP and T4polynucleotide kinase. Furthermore, fluorescent, chemoluminescent,spin-label, and redox reactive (i.e. for electrochemical detection)groups are commonly employed by those skilled in the art. One skilled inthe art would recognize that differential bias caused by theincorporation of such a label should be avoided. Thus, in someembodiments of the present invention each individual sequence member ofthe combinatorial pool is labeled identically. It should be furtherrecognized that the reporter group may contribute to the specificactivity of an oligonucleotide. Thus, the reporter group may be treatedas another monomer unit analog and permanently incorporated into thecomposition.

Unlabeled oligonucleotides determined according to the present methodsmay be sequenced, and this sequence information may be used toresynthesize labeled oligonucleotides. Labeled oligonucleotides may beuseful as probes or in other assays by virtue of their specific activityfor a target molecule and their ease of detection.

Oligonucleotides of the present invention may be of predeterminedlength. It is preferred that such oligomers be from about 6 to about 50units in length. It is more preferred that such oligomers be from about10 to about 20 units in length.

In some aspects of the present invention oligonucleotides may be insolution. In other aspects of the invention sequence randomizedoligonucleotides may be bound to solid support without sequencepreference.

Optimization of Sequence Determination

The term "random", as used herein, is used in the sense ofoligonucleotides having truly random base sequence, formed through solidstate synthesis. This method need not, however, be completely andstatistically random in fact. Thus, enrichment of oligonucleotides incertain bases may be desired in accordance with some embodiments of thisinvention. In the context of the present invention a randomoligonucleotide pool or library is a set of oligonucleotides havingsequences representing every possible combination of nucleotide unitswithin the randomized positions. Thus, in accordance with the presentinvention an oligonucleotide may have a complexity of P^(N) where P isthe number of different units used and N is the number of positions inan oligonucleotide which are randomized. For example, an oligonucleotidepool of oligonucleotides having six random positions comprising fourdifferent subunits would have a complexity of 4⁶ or 4096 differentsequences representing every possible combination of subunits. Ofcourse, a randomer pool may not necessarily be random in fact, andenrichment of oligonucleotides in certain bases may be desired inaccordance with some embodiments of this invention.

Unlike the preparation of randomized peptide libraries which have largedegrees of incorporation bias away from the molar ratio of the premixedunit members, Rutter and Santi, U.S. Pat. No. 5,010,175 issued Apr. 23,1991, the coupling efficiencies of natural nucleotide units and ofanalogs of limited chemical diversity using commercially availableautomated synthesizers and standard phosphoramidite chemistry known tothose skilled in the art appears to be very similar. That is, nearequimolar incorporation at a given randomized position of all monomerunit members of an equimolar mixture is obtained for randomizedlibraries of sufficient total quantity and limited complexity. Thus, inaccordance with preferred embodiments of the present invention, columnsof a DNA synthesizer may be packed with a mixture containing an equalamount of adenosine(A)-, cytidine(C)-, guanosine(G)- and uracil(U) (orthymidine(T))-controlled pore glass (CPG, Chemgenes, Needham, Mass.). Ateach "random" coupling step, an equimolar mixture of all fourphosphoramidites may be delivered to each column. Nucleic acidsynthesizers are commercially available and their use is generallyunderstood by persons of ordinary skill in the art to generate nearlyany oligonucleotide of reasonable length which may be desired. In somepreferred embodiments of the present invention the sequence randomizedoligonucleotides are cleaved from the controlled pore glass to provide apool of sequence randomized oligonucleotides in solution.

Randomized oligonucleotides made using monomer groups having greatersequence and structure complexity may require appropriate premixtureratio correction factors in order to obtain equimolar incorporation ofany monomer unit in the presence of any other monomer units atrandomized sequence positions of combinatorial libraries of the presentinvention. Strategies to determine coupling efficiencies and to correctfor any observed coupling bias are known to those skilled in the art.For example, Rutter and Santi, U.S. Pat. No. 5,010,175 issued Apr. 23,1991 and Lam, et al., PCT US91/06444 filed Jul. 1, 1991, describestrategies to accommodate incorporation bias in randomer peptidelibraries.

There is a complexity limit to the detectability of specific activity(signal to noise), especially in longer oligonucleotide librariescomposed of larger numbers of chemically distinct monomer units in whichthe oligonucleotides have a high percentage of randomized positions. Itis likely that with short, i.e. 8-10 nucleotides in length,unstructured, conformationally dynamic oligonucleotides, such as mightcommonly be used to initially limit complexity to facilitateidentification of highest specific activity sequences, a plethora ofrelatively weak specific activity towards many target molecules willresult. Procedures useful for increasing the complexity of anoligonucleotide pool and for increasing the specific activity ofoligonucleotide pool are encompassed by the present invention.

The use of additional units such as nucleotide analogs may be preferredin some instances where it is desirable to increase the complexity ofthe group of oligonucleotides, thereby increasing the specific activity.The complexity of a group of random oligonucleotides is represented bythe formula P^(N) where P is the number of different units used and N isthe number of positions in an oligonucleotide which are randomized.Table 1 illustrates the change in group complexity as a result of theincrease in the number of analogs used.

                  TABLE I                                                         ______________________________________                                        Oligonucleotide: NNNNNN                                                       Number of different                                                                          Complexity of each set (Q)                                     analogs used (P)                                                                             (P.sup.6)                                                      ______________________________________                                        4              4.sup.6 = 4096                                                 5              5.sup.6 = 15,625                                               6              6.sup.6 = 46,656                                               7              7.sup.6 = 117,649                                              8              8.sup.6 = 262,144                                              9              9.sup.6 = 531,441                                              10             10.sup.6 = 1,000,000                                           ______________________________________                                    

Initial screening protocols may be useful to determine whether a givenmonomer unit or analog will have a positive, negative or neutral effectin a given library pool for a pre-chosen target. Determination can bemade by incorporating equimolar amounts of all chosen monomer units atevery position of a pool and comparing the specific activity of the poolfor a target molecule to the specific activity of a combinatorial poolprepared without the monomer unit in question.

An additional method of increasing the specific activity of a group ofoligonucleotides is to constrain the oligonucleotide conformationally inorder to provide the correct presentation of the randomeroligonucleotide for the target biomolecule. There are two generalclasses of conformationally constrained chemistries, noncovalent andcovalent. Noncovalent bonding is generally easier as it can be achievedwithout additional chemistry steps. For example, an oligonucleotide maybe conformationally constrained by providing short complementary ends atthe 3' and 5' termini of the region of interest, which region ofinterest comprises randomized positions. Generally the complementaryends range in length from about 3 to about 8 nucleotides in length. Thecomplementary ends will hybridize to form secondary structure therebyproviding correct presentation of the randomized portion of theoligonucleotide to the target biomolecule. In accordance with otheraspects of the invention, noncovalent conformational constraints may beachieved with physiologic divalent metal bridge chelation of the 5' and3' ends of oligonucleotides.

Covalent constraints may also be provided by the incorporation ofcomplementary reactive groups at different fixed positions in theoligonucleotides of the combinatorial library. This may result inspontaneous unique covalent bond formation to form crosslinks, under theappropriate reaction conditions which will be apparent to those skilledin the art. Reactive groups and nucleotide analogs useful in theformation of crosslinking groups are described for example, by U.S. Ser.No. 846,376 filed Mar. 5, 1992 and incorporated by reference herein inits entirety.

Detectable specific activity may also be enhanced by a sequential methodin which evolutions of oligonucleotide pools, each of greater complexitythan its predecessors, are assayed for specific activity andcharacterized. This method allows selection in a randomized environmentwithin a complexity limit that is easily handled. Thus, for example,during one round of determination performed in accordance with methodsof the present invention an oligonucleotide specific for a targetbiomolecule is identified from a random pool of oligonucleotides.Thereafter, a next pool of longer oligonucleotides may be preparedcapitalizing upon the oligonucleotide sequence determined in theprevious step (i.e. a cassette) by incorporation of the determinedsequence or cassette into the sequence of the pool to provide a pool ofoligonucleotides having at least one cassette and at least one flankingregion of randomized positions. Thereafter the pool of oligonucleotidesis assayed for specific activity for a target biomolecule, and thesequence of the active oligonucleotide or oligonucleotides isdetermined. This procedure may be performed iteratively, building theoligonucleotides of each new round based upon the determined sequencesfrom the previous step or steps.

In some embodiments of the present invention it may also be desirable tosubfractionate a pool of sequence randomized oligonucleotides to providesubfractions of the oligonucleotide pool, thus limiting the degree ofcomplexity of each subfraction that is assayed at one time. This bothdiminishes the amount of total material that must be used in adetermination in order to have sufficient representation of allindividual sequences and it also enhances the signal to noise ratio ofthe assay by starting with an oligonucleotide subfraction enriched inthe most active sequences. Any physical-chemical or functionalcharacteristic, combined with an appropriate separation modality may beused to empirically subfractionate a group, thereby resulting in (orderiving) numerous distinct subfractions of diverse character, anddiminished complexity. It is theorized that if a sequence (or sequences)exists within the original group that have high affinity and specificityfor a particular target, it will be found enriched in a limited numberof the reduced complexity subfractions.

One skilled in the art would be apprised of the broad selection ofappropriate selection modalities which are available. The strategyfollowed will of course depend upon the properties of the elements ofthe oligonucleotide pool. It will further be appreciated by one skilledin the art that as the number of group elements increases and thestructural and chemical diversity enlarges, there will be a greaterselection of separation strategies leading to increased subfractionationcapacity. By way of example, it is envisioned that novel oligomers maybe resolved into subfractions by any one or a combination of size,positive or negative charge, hydrophobicity and affinity interactions.Many chromatographic and analytical instrumental methods are known tothose skilled in the art which may be effectively applied to theseparation strategies encompassed herein.

Targets

Target molecules of the present invention may include any of a varietyof biologically significant molecules. By biologically significant, oneskilled in the art would understand that molecules inherently related tosome biological function are intended. In addition, biologicalsignificance need not be limited to disease related significance, butmay also mean significance to, for example, the greater understanding ofbiological functions, or the greater ability to monitor or controlnormal biological function. Target molecules may be nucleic acid strandssuch as regions of DNA or RNA. Target molecules may also be proteins,glycoproteins, carbohydrates, lipids, toxins, steroids, drugs orcofactors. In some preferred embodiments of the present invention, saidtarget molecule is a protein such as an immunoglobulin, receptor,receptor binding ligand, antigen or enzyme and more specifically may bea phospholipase, tumor necrosis factor, endotoxin, interleukin,plasminogen activator, protein kinase, cell adhesion molecule,lipoxygenase, hydrolase or transacylase. In other preferred embodimentsof the present invention said target molecules may be important regionsof DNA or RNA of the human immunodeficiency virus, Candida, herpesviruses, papillomaviruses, cytomegalovirus, rhinoviruses, hepatitisviruses, or influenza viruses. In still further preferred embodiments ofthe present invention said target molecule is ras 47-mer stem loop RNA,the TAR element of human immunodeficiency virus, the gag-pol stem loopof human immunodeficiency virus (HIV), the RRE element of HIV, the HIVtat protein or the 5'-UTR of Hepatitis C virus. Still other targets mayinduce cellular activity. For example, a target may induce interferon.

In some aspects of the present invention, a target protein may beidentified based upon the fact that proteins bind to free aldehydegroups while nucleic acids do not. Thus, a sampling of proteins whichhave been identified as potential targets may be bound to solid supportshaving free aldehyde groups such as nitrocellulose filters. For example,up to 96 proteins may be bound in individual wells of a 96-wellnitrocellulose filter manifold. In some embodiments of the presentinvention sequential concentrations of protein may be tested todetermine the effect of lowering the protein target concentration.Thereafter, an identical detectably labeled oligonucleotide group may beincubated with each protein sample under binding conditions. Thepreparation of labeled oligonucleotide groups is described herein. Thesupport is washed and the presence or absence of binding is detectedwhereby binding indicates that the oligonucleotide group has specificactivity for a given protein. As will be apparent to one skilled in theart, methods of detection of binding will be dependent upon the labelused.

Detection

Specific activity of oligonucleotides of the present invention may bedetected by methods known to those skilled in the art. At least aportion of the random oligonucleotide pool will most preferably havespecific activity for a target molecule. The remaining oligonucleotidesmay be separated from the active oligonucleotides by methods such as aredisclosed herein. Appropriate assays will be apparent to one skilled inthe art and oligomer concentration, target molecule concentration, saltconcentration, temperature, buffer and buffer concentration may bealtered to optimize a particular system. Initial assay conditions shouldpreferably be compatible with further steps in the procedure. Forexample, high concentrations of salt or reagents which may interferewith binding should be avoided in some embodiments of the invention. Insome preferred embodiments of the present invention, binding conditionssimulate physiological conditions. In other preferred embodiments of thepresent invention binding occurs in a buffer of from about 80 mM toabout 110 mM sodium chloride and from about 10 to about 15 mM magnesiumchloride. Oligomers may also generally be assayed for catalytic orenzymatic activity.

Gel shift assays may be used to visualize binding of an oligomer to atarget molecule and separate bound from unbound oligonucleotides. Inaccordance with methods of the present invention, radiolabelled targetmolecule bound to oligomer of the present invention may be run on a gelsuch as a polyacrylamide gel. Bound target molecule has less mobilitythan unbound target molecule, and therefore will not migrate as far onthe gel. The radioactive label allows visualization of the "shift" inmobility by standard procedures for example, by means of autoradiographyor by using a phosphorimager (Molecular Dynamics). In other embodimentsof the present invention a gel shift assay may be performed wherein anunlabeled target molecule may be detected preferentially bound toradiolabeled oligonucleotide selected from the sequence randomizedoligonucleotide pool.

Streptavidin-biotin capture is another useful assay for the detection ofspecific activity and separation of active oligonucleotide from inactiveoligonucleotides. A target may be biotinylated prior to incubation witha radioactively labeled random oligonucleotide pool. Biotinylation oftarget is a well known procedure which may be accomplished through anumber of known procedures. For example, an RNA target may bebiotinylated using 5' kinase reactions. The oligonucleotide pool isthereafter incubated with the target and the target molecule is capturedon streptavidin-coated solid support. Consequently any oligonucleotidewhich is bound to the target will also be captured. Any of a broad rangeof solid supports known in the art could effectively be used in methodsof the invention. For example, streptavidin-coated solid supports areavailable commercially such as for example, streptavidin-coated magneticbeads available from Promega (Madison, Wis.) and streptavidin coatedmicrotitre plates (Covalink) available from NUNC (Raskilde, Denmark) orLabsystems (Marlboro, Mass.). The solid support may be washed and thereaction may be reequilibrated to further enrich the "winning" sequence.

Affinity mapping by enzymatic or chemical cleavage may be used to detectbinding site(s) for an oligonucleotide to a target molecule or preferredbinding site(s) for members of a pool of oligonucleotides, and determinethe sequence of the hybridization site(s) on said target molecule. Thismay be achieved by using chemical (e.g. permanganate) or enzymatic(single-strand specific RNases, e.g. RNase H, RNase CL3, RNase T1)cleavage. In accordance with methods of the present invention, targetRNA to which members of a random pool of oligodeoxyribonucleotides arebound is subjected to cleavage mapping by contacting with the cleavingagent. Only target bound to oligonucleotide will result in cleavage ofthe target at the site of heteroduplex formation. As a result, thepreferred hybridization site on the target RNA can be identified and itssequence determined.

Assays are not limited to detecting binding affinity but may also detectother desired activities such as biological responses such as catalyticor enzymatic activity. It should be recognized that functionalactivities are always mediated by prior binding interactions. Thus,selection for function is also selection for binding.

Positive functional selection is preferred in some embodiments of thepresent invention. For example, selection for catalytic function may beachieved by covalently coupling the moiety containing a scissile bond tobe bound and cleaved to a solid support with a noncleavable linker. Theother side of the moiety containing the said cleavable bond is coupledto one end of oligonucleotides of the combinatorial pool. The pool isthus covalently coupled to the solid support via the intermediatesubstrate containing the bond to be cleaved. Only those sequences in thepool capable of binding to the substrate and cleaving the only reactivebond present will release themselves from covalent capture on the solidsupport. These sequences may then be recovered from the solution phaseand characterized. In the alternative, only those sequences thatcatalyze bond formation will bind covalently to the solid support via areversible linker. The non-bound oligonucleotides may be washed away andthe reversible linker cleaved to release the active sequences. Solidsupports, noncleavable and reversible linkers and attendant conjugationchemistry are well known in the art.

Separation

Techniques such as continuous flow mass transport methodologies(Giddings, J. Calvin, Unified Separation Science, John Wiley and Sons,Inc., New York, 1991) may be also performed to identify activeoligonucleotides and separate active from inactive oligonucleotides. Anyof a broad range of methodologies which involve the use of continuous,differential mass transport flow via multiple competing equilibriabetween interacting species in a system with sufficient resolving powerare envisioned for use in effecting desired resolution. Suchmethodologies must preserve and take advantage selectively of k_(d) forthe tightest complexes being the slowest of all competing on and offrate constants. Furthermore, selected methodologies must achieve highstringency separation while enhancing enrichment of bound complexes.Optimally the amount of target molecule should be much greater than theamount of individual sequences, but should not be greater than theamount of the total number of oligonucleotide sequences in thecombinatorial library used. This formula will allow some limitedcompetition of all individual sequences for binding to the target, yetthere will be sufficient target to retain most of the preferredoligonucleotide. What will allow success is the provision of enoughresolving capacity and power to handle the relative and absoluteconcentrations and mass amounts of all components and the differentialaffinity of their interactions according to the performancecharacteristics of the particular systems. (Giddings, Unified SeparationScience, John Wiley and Sons, Inc., New York, 1991). Separationmethodologies dependent on nonspecific physical-chemical properties maybe employed. Preferred methodologies include those methodologies inwhich specific affinity interactions are utilized such as solid supportbased affinity chromatography wherein the target molecule is stablyattached to a solid support and the ligands are in the flowing mobilephase. High specificity affinity separation can also be achieved whenboth the target molecule and the ligand pool are in the mobile phase.Forces effecting differential transport of bound target and free ligandsis all that is required. Resolution may also be enhanced by theinclusion of a mass excess (over the randomized oligonucleotide ligandpool) of a uniformly nonspecific, weak binding molecule (such as bovineserum albumin) of the ligand pool that does not interact with the targetmolecule.

Representative resolution modalities which may be useful in methods ofthe present invention include, but are not limited to, sizeexclusion/gel filtration chromatography (Sephadex-LPLC orSuperous-FPLC), isoelectric focusing (IEF) including Rotofor IEF(Bio-Rad), fast affinity chromatography (Beckman), electrophoresismethods, sedimentation methods, field flow fractionation (FFF), ionexchange chromatography, weak affinity chromatography; Zopf and Ohlson,Nature, 1990, 346, 87; affinity filtration, centrifugal countercurrentchromatography (CCC), perfusion chromatography, affinity chromatographyon solid supports, and hydrophilic interaction chromatography.

Microanalytical resolution techniques may also be employed to separateactive from inactive oligonucleotides. In some embodiments of thepresent invention microanalytical separation techniques may also beuseful to resolve a group of active oligonucleotides. Microanalyticalresolution technologies are well known in the art and any of thetechnologies will be useful in the present invention. For example, gaschromatography (gc), high Performance liquid chromatography (HPLC), highperformance capillary electrophoresis (HPEC), and field flowfractionation (FFF), (Giddings, J. Calvin, Unified Separation Science,John Wiley and Sons, Inc., New York, 1991), may be used alone or on-linewith microanalytical characterization technologies. HPLC methods haveevolved to accommodate the resolution of nanomole and lesser quantitiesof materials. For example, microbore HPLC on-line with mass spectrometryhas been applied to resolve a combinatorial problem. Hunt, et al.,Science, 1992, 255, 1261. HP capillary electrophoresis (HPCE) may alsobe effectively applied to resolve oligonucleotides. When HPCE is placedon-line with mass spectrometry there is no requirement to be able todirectly resolve sequences prior to injection in the mass spectrometer.Use of this methodology is described in Toulas, et al., LC-GC, 1992, 10,471 (detection of attomolar to zeptomolar amounts of fluorescencereporter group tagged oligonucleotides with laser-induced fluorescencedetection); Rodrigues, et al., Amer. Biotech. Lab. 1992, 21, (capillarynon-gel sieving electrophoresis of nucleic acids); Carchon, et al.,Amer. Biotech. Lab. 1992, 67; Stevenson, Amer. Lab, 1992, 17; Stevenson,Chem. and Engin. News, 1992, 24, (micellar electrokinetic capillaryelectrophoresis); Cobb, et al., Anal. Chem. 1992 (mapping and sequencedetermination of pre-fragmented oligomers from HPCE). Two dimensionalelectrophoresis of oligonucleotides may also be useful, especially whereoligonucleotides are comprised of chemically diverse monomers.

In accordance with certain embodiments of the present invention activeoligonucleotides are recovered. Recovery of active oligonucleotides, asused herein, refers to sufficient purification of the activeoligonucleotide(s) for performance of subsequent steps. Thus, in someaspects of the invention that recovery of active oligonucleotides refersto separation of active oligonucleotides from target biomolecule. Insome preferred embodiments of the present invention activeoligonucleotides may be recovered attendant to separation of the activefrom inactive oligonucleotide. In other embodiments of the presentinvention, recovery requires additional steps. In still otherembodiments of the present invention, only the binding ofoligonucleotides to the target molecule may be determined, in which caserecovery of the oligonucleotide may not be necessary.

Characterization

Active oligonucleotides may be characterized, such as by microanalyticalcharacterization or standard nucleic acid sequencing. In some preferredembodiments of the present invention, separation, recovery, andcharacterization are accomplished by the use of microanalyticalresolution technologies combined with microanalytical characterizationtechnologies, performed individually or integrated such as in "on-line"systems for maximum efficiency and conservation of material.

The microanalytical technique mass spectrometry (MS) may be effectivelyused to characterize oligonucleotides. MS has been successfully appliedto peptides. For example, Hunt, et al., Science, 1992, 255, 1261 and1264 successfully identified the sequences of peptides (with theoreticalcomplexity of 20⁹) selected for binding (intracellularly) to MHCreceptor molecules. After recovery and crude purification away fromcontaminating cellular components, the preferred peptide-MHC complexes(>200) were dissociated and the peptides resolved by microcapillary HPLCon-line to electrospray ionization tandem quadrupole FAB-MS, whichidentified the sequences of eight of the peptides present in onlysub-pmole amounts.

MS can also be applied to both fully and partially resolvedoligonucleotides. Furthermore, new non-destructive volatilizationprocedures now allow for MS application to biopolymers and analogs. Forexample, electrospray ionization (Weinberger, Amer. Lab. 1992, 54; andlaser desorption, (Romano and Levis, J. Amer. Chem. Soc., 1991, 113,9665.

In other preferred embodiments of the present invention oligonucleotidesare characterized by amplification and sequencing. Thus, in accordancewith the present invention, a polynucleotide tail such as polyA is addedto the 3' end of the highest specific activity selectedoligonucleotide(s) to form a first strand. The term polyA is used torefer herein to the addition of a riboadenosine (to tail an RNAmolecule) or deoxyadenosine (to tail a DNA molecule) and may be addedvia terminal deoxynucleotidyl transferase (TdT) for DNA or polyrApolymerase for RNA. Other methods of providing polynucleotide tailsknown to those skilled in the art are also encompassed herein. A firstchimeric primer is hybridized to the first strand. The first chimericprimer is comprised of a 5' known sequence and a 3' polynucleotideportion complementary to the polynucleotide tail of the first strand.Thus, in the present example the first chimeric primer is comprised of aknown sequence and a 3' polydT portion. The 3' polynucleotide portion ispreferably approximately from about 12 to about 16 nucleotides, and mostpreferably 15 nucleotides in length in order to optimize hybridizationof the primer to the polynucleotide region of the first strand at lowertemperatures. The 5' known sequence is preferably approximately 10 toabout 15 nucleotides in length, and most preferably 12 nucleotides inlength. The 5' known sequence is preferably also comprised of a high GCcontent that together with the 3' polynucleotide region allows forstringent binding at elevated temperatures. Thus, at higher temperaturesrequired for PCR, there should be no cross-priming of the polynucleotidetails. In still further preferred embodiments of the present invention,the 5' region of the first chimeric primer may provide for one or moreunique restriction sites. Such restriction sites may be useful forexample, in forced cloning. Furthermore, the unique sequence serves abookkeeping purpose in order to discriminate the sense from theantisense sequence in final sequencing. In yet further embodiments ofthe invention the first chimeric primer provides for mechanisms wherebythe primer may be excised from the oligonucleotide of interest. Forexample, the first chimeric primer may incorporate a class IISrestriction endonuclease site. Class IIS restriction endonucleases("shifters") cleave double stranded DNA at a precise number ofnucleotides from the recognition site, regardless of the sequence ofthose nucleotides. For example, FokI cleaves double stranded DNAthirteen nucleotides from its recognition site, thereby removing theregion downstream from the oligonucleotide of interest.

In still more preferred embodiments of the present invention a binding"handle" may be provided as a reactive group incorporated into the firstchimeric primer. For example, a biotin moiety may be incorporated duringsynthesis at the 5' end of the first chimeric primer. Other binding"handles" will be apparent to one skilled in the art given the presentdisclosure.

In certain embodiments of the present invention, the first chimericprimer may be captured on a solid support. For example, the primer maybe streptavidin captured using an incorporated biotin binding "handle".In other embodiments of the present invention the primer may becovalently attached to microtitre plates such as by modifications of themethod described by Mitsuhashi, et al., Nature,1992, 357, 519. Suchcapture may facilitate washing during the preparation of the flanking"primer sites" flanking either end of the oligonucleotide of interest.

Polymerase such as Taq I polymerase in the presence of an excess of allfour nucleotides may be used to form cDNA complementary to the firststrand using the first chimeric primer. The cDNA may be further extendedat its 3' end with a polynucleotide tail such as polydA by addition ofexcess nucleotide in the presence of an appropriate enzyme such as TdT.For example, the cDNA may be further extended at its 3' end with apolydA by the addition of dATP in the presence of TdT. The first strandand the cDNA strand may subsequently be separated by heating.

A second chimeric primer may then be hybridized to the cDNA strand at alower hybridization temperature, resulting in 3' recessed ends on bothstrands. The second chimeric primer, like the first, is comprised of a5' known sequence and a 3' polynucleotide portion which is complementaryto the polynucleotide tail of the cDNA. Thus, in the present example,the second chimeric primer has a 3' polydT portion which iscomplementary to the polyA tail of the cDNA. The 3' polynucleotideportion is preferably approximately from about 12 to about 16nucleotides, and most preferably 15 nucleotides in length and the 5'known sequence is preferably approximately 10 to about 17 nucleotides inlength, and most preferably 13 or 15 nucleotides in length. The 5' knownsequence of the second chimeric primer is also comprised of a high GCcontent that together with the 3' polynucleotide region allows forstringent binding at elevated temperatures and may provide for a uniquerestriction site. The second chimeric primer also provides formechanisms whereby the primer may be excised from the oligonucleotide ofinterest. For example, the second chimeric primer may incorporate ariboU at the 3' terminus. The ribophosphate diester bond may be cleavedby selective specific base (--OH) hydrolysis or RNase treatment, thusexcising the primer and any upstream regions from the region downstreamfrom the ribou site.

The recessed ends are filled in by the addition of a polymerase such asTaq I and an excess of all four nucleotides. Thus, using recessed endsas primers and the overhangs as templates, complementary strands areformed. The resulting fully complementary and fully duplex strands areseparated by heating and polymerase chain reaction is performed toamplify the oligonucleotides. Polymerase chain reaction procedures arewell known in the art. Ausubel, et al., Current Protocols in MolecularBiology, John Wiley and Sons, 1989. The first and second chimericprimers may effectively serve as PCR primers.

The amplified oligonucleotides may be force cloned using the primersequence unique restriction sites or PCR product single base overhangcloned and sequenced by procedures known in the art. Alternatively, insome embodiments of the present invention the oligonucleotide ofinterest may be excised from the upstream and downstream flankingregions, re-assayed for selective binding, recovered and characterized.For example, by relying upon mechanisms incorporated within primershaving the sequences GGATG(dT)₁₃ (SEQ ID NO: 1; first chimeric primer)and CGC TGG ATC CGC (dT)₁₄ rU (SEQ ID NO: 2; second chimeric primer) theoligonucleotide may be restricted with FokI, or the ribophosphatediester bonds may be selectively cleaved by hydrolysis or treatment withRNase to excise the original random oligonucleotide of interest from theflanking regions added herein to facilitate amplification of theoligonucleotides.

In other embodiments of the present invention, the sequence of an activeoligonucleotide (one bound to a target molecule) may be determined byidentifying the preferred binding site of the oligonucleotide to thetarget molecule. This can be followed by optimizing the binding ofoligonucleotide to the target molecule. Oligonucleotides which optimallytarget the sequence and structure of a target biomolecule provideincreased affinity and specificity, and thereby enhanced specificactivity for that biomolecule. In one embodiment, methods of the presentinvention have been used to determine preferred hybridization sites on atarget RNA molecule by means of affinity mapping using enzymatic orchemical cleavage techniques. Random oligonucleotide pools are mixedwith the target molecule and allowed to hybridize. Stringent selectionconditions are enlisted to drive the selection in favor ofoligonucleotides that exhibited the highest affinity for the target RNA.This end is achieved by allowing hybridization to proceed underconditions wherein the concentrations of the individual oligonucleotidesof the pool were much lower than the concentration of the target RNA.The location(s) of bound oligonucleotide(s) is then mapped usingenzymatic or chemical cleavage. In one preferred embodiment, RNase H isused. RNase H is a ribonuclease that cleaves the RNA portion of aDNA:RNA hybrid, and thus, can be used to map the location of boundoligonucleotides on the target RNA. In a preferred embodiment, affinitymapping is followed by optimization of the oligonucleotide sequence andlength for binding to these sites on the target RNA with a quantitativefootprinting assay, which reveals the tightest binding oligonucleotidesas indicated by the lowest K_(d) values. This allows determination ofthose oligonucleotides that exhibit best binding to the preferred targetsite. A series of oligonucleotides are prepared which comprise asequence complementary to at least a portion of the preferred targetsite identified by cleavage mapping. In a preferred embodiment, a set ofoligonucleotides with overlapping sequences is prepared.

The affinity of each oligonucleotide for the target is determined, and aK_(d) for each is determined. The oligonucleotide with the lowest K_(d)is the "winner," i.e. demonstrates optimal binding. K_(d) can bedetermined by gel shift analyses or by footprinting. In a more preferredembodiment, RNase ONE is used for footprinting. RNase ONE is abase-independent, single strand-specific endoribonuclease. In otherembodiments, single-strand specific RNases such as RNase T1, RNase CL3or other enzymatic cleavers, or chemical cleaving agents such aspermanganate are used. Oligonucleotides identified by this strategy havebeen shown to be functionally active inhibitors of target RNAexpression.

The oligonucleotides of the present invention can be used indiagnostics, therapeutics and as research reagents. For therapeuticuses, an effective amount, ranging from 1 pg/kg to 1 g/kg of bodyweight, of the oligonucleotide is administered, to an animal, especiallya human, suffering from a disorder effected by a biomolecule. Saidbiomolecule may be derived from an infectious agent such as aherpesvirus, or other viruses. Further, said biomolecule may be derivedfrom a noninfectious agent of, for example, genetic origin. The regimenof administration may vary from once a day to several times a day, andmay also be modified so as to administer the oligonucleotide severaltimes a year or once in several years, depending on the degree ofresponse observed. Effective amounts of therapeutic agents of thepresent invention, may be applied topically, intralesionally, orally,transdermally, intravenously or intramuscularly, as appropriate for theparticular disorder to be treated. One skilled in the art would beapprised of the method most effective for any given disorder. Use ofpharmacologically acceptable carriers is also preferred for someembodiments.

The oligonucleotides of the present invention may be used to detect anddistinguish between two ligands, such as small, structurallyclosely-related molecules. For example, Jenison, et al., Science, 1994,263, 1425, demonstrate the ability of RNA molecules to display a highdegree of molecular recognition and discrimination between theophyllineand caffeine. Their results demonstrate the utility of oligonucleotidesas diagnostic agents. Oligonucleotides and methods of the presentinvention are also useful in research as they may be easily adapted tosuit a range of assays. Oligonucleotides and methods of the presentinvention are also useful in therapeutics, for example, in processessuch as pheresis, as highly specific sequestering agents for excess drugmolecules, in the case of overdose. Thus, in accordance with methods ofthe present invention, oligonucleotides identified by methods of thepresent invention as being specific to a particular target molecule suchas a drug or chemical, can be used to detect the chemical or drug in asample such as a sample consisting of cells, tissues or bodily fluids,by contacting the sample with the oligonucleotide and detecting thepresence or absence of binding by the oligonucleotide. The presence ofbinding is indicative of the presence of a specific chemical or drug inthe sample. Binding assays are well known in the art. In preferredembodiments, the oligonucleotide is detectably labeled to facilitatedetection of binding.

The following examples are illustrative, but not limiting of the presentinvention.

EXAMPLE 1

Synthesis of DNA Oligonucleotides

Unmodified DNA oligonucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380 B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl phosphoramidites may be purchased from AppliedBiosystems (Foster City, Calif.).

EXAMPLE 2

Synthesis of RNA Oligonucleotides

Unmodified RNA oligonucleotides having random base sequences weresynthesized on an automated DNA synthesizer (Applied Biosystems model380 B) using modified standard phosphoramidite chemistry synthesis withoxidation by iodine. The standard synthesis was modified by increasingthe wait step after the pulse delivery of tetrazole to 900 seconds.β-cyanoethyldiisopropyl phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). The bases were deprotected byincubation in methanolic ammonia overnight. Following base deprotection,the oligonucleotides were dried in vacuo. The t-butyldimethylsilylprotecting the 2' hydroxyl was removed by incubating the oligonucleotidein 1 M tetrabutylammoniumfluoride in tetrahydrofuran overnight. The RNAoligonucleotides were further purified on C₁₈ Sep-Pak cartridges(Waters, Division of Millipore Corp., Milford, Mass.) and ethanolprecipitated.

EXAMPLE 3

Synthesis of Phosphorothioate Oligonucleotides

Phosphorothioate oligonucleotides represent a class of oligonucleotideanalog that is substantially nuclease resistant. Phosphorothioate RNAoligonucleotides and phosphorothioate DNA oligonucleotides weresynthesized according to the procedure set forth in Examples 1 and 2respectively, replacing the standard oxidation bottle by a 0.2 Msolution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forstepwise thiation of phosphite linkages. The thiation cycle wait stepwas increased to 68 seconds and is followed by the capping step.

EXAMPLE 4

Synthesis of 2'-O-alkyl Phosphorothioate Oligonucleotides

2'-O-methyl phosphorothioate oligonucleotides were synthesized accordingto the procedures set forth in Example 3 substituting 2'-O-methylβ-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham, Mass.) forstandard phosphoramidites and increasing the wait cycle after the pulsedelivery of tetrazole and base to 360 seconds. Similarly, 2'-O-phenyl,2'-O-propyl and other 2'-O-alkyl phosphorothioate oligonucleotides maybe prepared by slight modifications of this procedure.

EXAMPLE 5

Preparation of Pyrene Oligonucleotide Analogs

Oligonucleotides were prepared by incorporating 2' aminopentoxyadenosineat desired sites. The oligonucleotides were dissolved in 0.2 M NaHCO₃buffer and treated with 50-fold excess of N-hydroxysuccinimide ester ofpyrene-1-butyric acid dissolved in dimethylformamide. The resultantmixture is incubated at 37° C. for 4-5 hours and the conjugate ispurified by reverse phase HPLC followed by desalting in a G-25 Sephadexcolumn.

EXAMPLE 6

Synthesis of Oligonucleotide Pools Having Randomized Positions

Four columns of the DNA synthesizer were packed with a mixturecontaining an equal amount of adenosine(A)-, cytidine(C)-, guanosine(G)-and thymidine(T)- or uridine(U)-controlled pore glass (CPG, Chemgenes,Needham, Mass.). At coupling steps where a given nucleotide base wasdesired, the defined phosphoramidite was delivered to each column. Ateach "random" coupling step, an equimolar mixture of all fourphosphoramidites was delivered to each column.

EXAMPLE 7

Preparation of Radiolabeled Groups

oligonucleotide groups prepared in accordance with Example 1 through 6are radiolabeled using [γ³² P] ATP and T4 polynucleotide kinase asdescribed in Sambrook et al., Molecular Cloning. A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 11.31-11.32.

EXAMPLE 8

Preparation of Oligonucleotide Group Comprising Nucleotide Analogs

Oligonucleotide groups are prepared in accordance with Example 1 and 6incorporating one or more of the nucleoside analogs 2'-O-nonyladenosine, 6-N-imidazoylpropyl guanosine, 2'-O-aminopentyl cytidine,2'-O-pentyl-adenosine, 2'-O-pentyl-guanosine, 2'-O-pentyl-cytidine,3'-terminal 2'-O-methyl uridine and 6-amino-2-hydroxylmethyl-1-hexanol.The nucleosides, 2'-O-nonyl adenosine, N6-imidazoylpropyl guanosine,2'-O-aminopentyl cytidine, 2'-O-pentyl-adenosine, 2'-O-pentyl-guanosine,2'-O-pentyl-cytidine, 3'-terminal 2'-O-methyl uridine were prepared bymodification of the methods described in PCT US91/00243 filed Jan. 11,1991. 6-amino-2-hydroxylmethyl-1-hexanol is available commercially. Thenucleosides are modified to provide the corresponding phosphoramidite bymethods known to those skilled in the art.

EXAMPLE 9

Gel Shift Assay of Random DNA Oligonucleotide Binding to ras RNA Targetand Enzymatic Amplification of Active Oligonucleotides

The ras 47-mer stem/loop RNA was enzymatically synthesized, ³² Pend-labeled according to standard procedures, and gel-purified. The rastarget is incubated at a concentration of approximately 10-50 pM withDNA oligonucleotide pools synthesized in accordance with the methoddescribed in Examples 1 and 6 at concentrations of 1, 5, 10, 50 and 100μM in a buffer consisting of 100 mM NaCl and 10 mM MgCl₂. Thehybridization is carried out for four hours at 37° C., followed byelectrophoresis separation of bound vs. unbound material on a 20%polyacrylamide gel in Tris-Borate buffer (TBE) plus 50 mM NaCl run at 25W for four hours. The gel is dried and the radioactive bands arevisualized on a phosphorimager (Molecular Dynamics). The ras stem/looptarget alone will be the lowest band visible on the gel (highestmobility). As this target binds oligonucleotide (non-radioactive), themobility of the ras target will decrease, shifting the band to a higherposition on the gel (complex). The bound complex is excised from the gelwith a sterile razor blade. The oligonucleotides (ras RNA and boundoligonucleotide(s)) are recovered by the crush and soak method. The rasRNA and binding selected oligonucleotide(s) are separated by size usingHPLC procedures and the fraction(s) containing the binding selectedoligonucleotides collected. The recovered oligonucleotides are amplifiedin accordance with the following steps:

(1) Poly dA tailing of recovered oligonucleotides to form a first strand

Tailing of recovered oligonucleotides is carried out in a microtitrewell using 5-50 units terminal deoxynucleotide transferase (BoehringerMannheim Biochemicals) according to manufacturer's instructions [2hours, 37° C. in tailing buffer (supplied with enzyme) plus 1.5 mM CoCl₂and 100 nM dATP]. Reaction volume is 20 μl.

(2) Preparation of a first chimeric primer

A first chimeric primer is synthesized by automated chemical synthesis.At the 5' end is a known sequence incorporating a ribonucleaserestriction site for FokI and a biotin moiety at the 5' termini. At the3' end is a 13 nucleotide polydT stretch. This primer is added atmicromolar concentrations to the reaction mixture in a buffer consistingof 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and allowed to hybridize to thefirst strand.

(3) Capture of the first chimeric primer with 5'-biotin-streptavidin

A microtitre well is coated with 50 μg/ml streptavidin in sodiumcarbonate, pH 9.25 for 3 hours at 37° C. Alternatively, commerciallyavailable streptavidin ready-coated microtitre plates (Labsystems,Marlboro, Mass.) can be used. Nonspecific binding sites are blocked with0.3% bovine serum albumin in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl at 37°C. for 3 hours. The first chimeric primer/oligonucleotide duplex is thenbound to the streptavidin-coated plates by incubating for 1 hour at 37°C. in 100 mM Tris-HCl, 150 mM NaCl, pH 8.0.

(4) Polymerase copy of first strand to form double-stranded duplex:

To the microtitre well is added 0.4 units Taq polymerase (Promega),micromolar amounts of each dNTP, and 1×polymerase buffer (Promega) to afinal volume of 50 μl. The reaction is carried out for 3 to 12 hours atroom temperature. Well are washed six times in wash buffer.

(5) Poly-dA tailing of cDNA of duplex

Tailing of the cDNA is carried out in a microtitre well using 5-50 unitsterminal deoxynucleotide transferase (Boehringer Mannheim Biochemicals)according to manufacturer's instructions [2 hours, 37° C. in tailingbuffer (supplied with enzyme) plus 1.5 mM CoCl₂ and 100 nM dATP]. Wellis washed 6 times with 200 μl of wash buffer.

(6) Separation of cDNA and oligonucleotide

The microtitre plate is heated to 85° C. to melt the duplex and thesupernatant is removed. The well is washed six times with 200 μl washbuffer.

(7) Preparation of a second chimeric primer

A second chimeric primer is synthesized by automated chemical synthesis.At the 5' end is a known sequence incorporating a ribonucleaserestriction site for BamH1. At the 3' end is a 14 nucleotide polydTstretch ending in a riboU at its termini. This primer is added atmicromolar concentrations to the reaction mixture in a buffer consistingof 10 mM Tris-HCl, pH 7.5, 150 mM NaCl and allowed to hybridize to thefirst strand.

(8) Filling in recessed ends to form a duplex

To the microtitre well is added 0.4 units Taq polymerase (Promega),micromolar amounts of each dNTP, and 1×polymerase buffer (Promega) to afinal volume of 50 μl. The reaction is carried out for 3 to 12 hours atroom temperature. Wells are washed six times in wash buffer. Themicrotitre plate is heated to a 85° C. to melt the duplex.

(9) PCR Amplification of Oligonucleotides

PCR amplification is performed using Taq polymerase according tostandard methods (Frederick M. Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, 1989) using first and secondchimeric primers from (2) and (7) as PCR primers.

(10) Excising oligonucleotide from upstream and downstream flankingregions

Amplified oligonucleotides are excised from flanking regions by cleavagewith FokI in accordance with suppliers recommendations (BoehringerMannheim). After FokI restriction and melting apart of the restrictedsense and antisense fragments, the reaction mixture was treated withmild base to cleave the upstream primer region from the sense strand byselective hydrolysis of the ribophosphate diester linkage between the3'-riboU of the primer and the 5' region of the oligonucleotide ofinterest.

(11) The selection steps and amplification steps are repeated until onlyone, or at most only a few, unique oligonucleotide sequencesreproducible are recovered from "round to round".

(12) Sequencing of amplified oligonucleotide(s):

The sequence is determined using the standard Sanger ddNTP method andSequenase enzyme (Pharmacia, Inc.) (Frederick M. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, 1989). Sequencingcan be done with one, two, three, or all four ddNTPs, depending on theextent of sequence bias, or if exact sequence is desired.

EXAMPLE 10

Gel Shift Assay of Random 2'-O-Methyl Oligonucleotide Binding to ras RNATarget and Microanalytical Characterization

The ras 47-mer stem/loop RNA was enzymatically synthesized, ³² Pend-labeled according to standard procedures, and gel-purified. The rastarget is incubated at a concentration of approximately 10-50 pM withrandom 2'-O-methyl oligonucleotide pools synthesized in accordance withthe method described in Examples 4 and 6 at concentrations of 1, 5, 10,50 and 100 μM in a buffer consisting of 100 mM NaCl and 10 mM MgCl₂. Thehybridization is carried out for four hours at 37° C., followed byelectrophoresis separation of bound vs. unbound material on a 20%polyacrylamide gel in Tris-Borate buffer (TBE) plus 50 mM NaCl run at 25W for four hours. The gel is dried and the radioactive bands arevisualized on a phosphorimager (Molecular Dynamics). The ras stem/looptarget alone will be the lowest band visible on the gel (highestmobility). As this target binds oligonucleotide (non-radioactive), themobility of the ras target will decrease, shifting the band to a higherposition on the gel (complex). The bound complex is excised from the gelwith a sterile razor blade. The oligonucleotides (ras RNA and boundoligonucleotide(s)) are recovered by crush and soak method or byelectroelution. The ras RNA and binding selected oligonucleotide(s) areseparated by RNase degradation which selectively degrades the ras RNA tomononucleotides, leaving the selected, RNase resistant 2'-O-methyloligonucleotide(s) intact. The selected oligonucleotides are identifiedby the use of microbore HPLC and mass spectrometry to determine thebound oligonucleotide sequences. To facilitate MS sequencingdetermination, the recovered 2'-O-methyl oligonucleotide(s) can befragmented by prolonged treatment (compared to RNA or DNA) with any of anumber of available nucleases; sequencing of shorter fragments by MS iseasier and the sequence of the parent oligonucleotide can bereconstructed from overlapping sequences of the fragments.

EXAMPLE 11

Gel Shift Assay of Random Pyrene Oligonucleotide Sets Binding to HIV TARElement

The HIV TAR element is a structured RNA found on the 5'-end of all HIVtranscripts. A gel shift is used to analyze the binding ofoligonucleotides of a random oligonucleotide pool prepared in accordancewith Examples 5 and 6, each oligonucleotide pool containing a pyreneanalog to the HIV TAR element. The target RNA has a three base bulgethat is required for binding of the transcriptional activation proteintat. The assay uses a very low concentration of pools. Binding ofmolecules from the pool to the target results in a slower mobilitycomplex. Recovery of the bound oligonucleotide(s) and identification ofsequences can be accomplished as described in Example 10.

EXAMPLE 12

Random 2'-O-Methyl Oligonucleotide Binding to ras RNA Using ContinuousFlow Mass Transport Methodology to Effect Stringent Binding Selection

The ras 47-mer stem/loop RNA was enzymatically synthesized, ³² Pend-labeled according to standard procedures, and gel-purified.

A fast protein liquid chromatography (FPLC, Pharmacia) system iscalibrated to separate bound from unbound oligonucleotides. The columnused is a Superous 12 HR 10/30, fractionation MW range of 10³ -3×10⁵(for proteins), >40,000 theoretical plates/m, 1.0×30 cm, with ≦6-30mL/min flow rate. Starting conditions are 10-50 mM TE buffer, pH 7.0, 1mM magnesium chloride and 100 mM sodium chloride.

Calibration 2'-O-methyl oligonucleotides having the sequences UUGCCCACAC(SEQ ID NO:3), UUGCCCACAU (SEQ ID NO:4), UUGCUCACAC (SEQ ID NO:5),CUGCCCACAU (SEQ ID NO:6), and CUGUUUACAU (SEQ ID NO:7) are prepared andradiolabeled using polynucleotide kinase and ³² P-ATP. Alternatively,rather than label the oligonucleotides, the target molecule, ras RNA maybe ³² P labeled or both oligonucleotide and target may be labeled. Thesecalibration oligonucleotides should be of the same length as therandomized oligonucleotides to be tested and are of varying degrees ofsequence complementarity to the target molecule.

The FPLC column is calibrated before antisense selection fromcombinatorial pools by running the calibration ³² P labeled-2'-O-methyloligonucleotides; individually (for unbound species retention times(RT)) and as a mixture of calibration 2'-O-methyl oligonucleotides whichhas been incubated with ras RNA to form any possible hybridizationcomplexes (for retention times of bound and differential ratedissociated species). The mixture is then loaded on the column and runto provide an elution profile. Alternatively, the calibrationoligonucleotides may be added to the column as a mixture, followed bythe addition of the ras RNA. As the ras RNA should flow faster than theoligonucleotides, it should flow past the oligonucleotides. Thiscontinuous flow combining binding and resolution may allow for betterseparation and recovery because the mass action bulk equilibriumstarting point of pre-binding is to some extent avoided. The effect ofadding a weak, nonspecific binder of oligonucleotides in mass excess inthe mobile phase can similarly be assessed.

Following calibration, either the random oligonucleotide pool is loadedonto the column followed by the ras RNA or the pool and ras RNA arepre-incubated (37° C., 1-4 hours) prior to loading on the column. Boundoligonucleotide/ras RNA complex(es) are dissociated using stepwise orgradient low salt and/or increased temperature and the oligonucleotidesare recovered by RNAse treatment to selectively degrade the ras RNA. Theselected 2'-O-methyl oligonucleotides are characterized by microboreHPLC (Smart Systems from Pharmacia). Complete and limited fragmentationof the recovered 2'-O-methyl oligonucleotides can be accomplished byappropriate base and nuclease treatment to facilitate sequencereconstruction in comparison to pre-calibrated retention times ofstandard mono, di, and tri 2'-O-methyl standards.

EXAMPLE 13

FPLC Resolution of Random RNA Oligonucleotide Binding to ras RNA

The ras 47-mer stem/loop RNA is enzymatically synthesized, isbiotin-labeled according to standard procedures, and gel-purified.Calibration oligonucleotides were prepared and FPLC system calibrated asdescribed in Example 12. An RNA random oligonucleotide pool was preparedin accordance with Examples 2 and 6 and the FPLC binding assay wasperformed as described in Example 12. The RNA-oligonucleotide(s) boundcomplex(es) were recovered using biotin-streptavidin capture of thebiotinylated ras RNA. The bound oligonucleotide(s)/ras RNA complex(es)are dissociated using low salt and/or increased temperature and theselected oligonucleotide(s) recovered from the supernatant. Theoligonucleotide was amplified according to a modification of theprocedure in Example 9.

EXAMPLE 14

In Vitro Evolution of Longer Oligonucleotides of Greater Complexity forEnhanced Binding Affinity and Specificity

A "winner" sequence is determined in accordance with one of the Examples9-13. The "winner" sequence is designated a cassette for purposes of theinvention, i.e. an oligonucleotide sequence determined by an initialapplication of methods of the invention.

In subsequent application of methods of the invention a randomizedoligonucleotide pool is prepared in accordance with Example 1 comprisingthe previously determined cassette and at least one randomized flankingregion. Selection for activity is performed and the oligonucleotidecharacterized as described in Examples 10-14. These steps can beperformed iteratively. Thus, the final sequence may subsequently be usedas a cassette in order to expand upon the known desired sequence tooptimize selective activity.

EXAMPLE 15

Identification of Oligonucleotide Sequence Using Streptavidin Capture ofBiotinylated Target

A target oligonucleotide (0.2 μM) having the sequence 3'dBAB AGA CGT CTTGCG 5' (SEQ ID NO:8) wherein B is biotin, was incubated for 30 minutesat room temperature with a radiolabeled 2'-O-methyl oligonucleotidesequence randomized pool (10 μM) having the sequence NNNNCNCNN wherein Nis any of adenine, cytosine, thymine or guanine. The targetoligonucleotide was captured on streptavidin-coated magnetic beads(Promega), the beads were washed, and supernatant removed. Thebead-containing sample was run on polyacrylamide gel to determine thesample having the highest binding. The binding selective steps wererepeated until a winner sequence was identified. Enrichment in each step(measured by radioactivity) was 200- to 1000-fold. Confirmation of thesequence of the final, best binding oligonucleotide was provided bycomplete blocking of binding of radioactivity by 0.1 μM of unlabeledtarget complementary to the sequence competitor.

EXAMPLE 16

Identification of a Protein Target

A group of oligonucleotides having the sequence NNNNNNNN wherein N isany one of adenine, guanine, thymidine or cytosine is prepared inaccordance with Example 3. The group is labeled using [γ³² P] ATP and T4polynucleotide kinase.

In individual wells of a 96-well nitrocellulose filter manifold, thefollowing proteins are incubated in a solution of phosphate bufferedsaline: plasminogen activator A₂, tumor necrosis factor α, tumornecrosis factor β and gp120. Phosphate buffered saline only is added toa control well. The filter is washed. An aliquot of the labeled group ofoligonucleotides is added to each well and incubated at room temperaturefor 10 minutes. The filter is washed and the counts in each well overbackground are counted to determine whether binding of theoligonucleotide to the protein occurred.

EXAMPLE 17

RNase H Affinity Mapping of ras RNA by RNAse H Cleavage Mapping

The ras 47-mer stem/loop RNA was enzymatically synthesized, ³² Pend-labeled according to Sambrook et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 11.31-11.32, and gel-purified. The ras target was incubated at aconcentration of approximately 1 μM to 10 nM with random oligonucleotidepools synthesized in accordance with the method described in Example 1at individual oligonucleotide concentrations of 100 pM in 10 mM Trisbuffer (pH 8) containing 50 mM NaCl and 5 mM MgCl₂. The hybridizationwas carried out for at least 16 hours at 37° C. RNase H (1 U/μL) wasadded in 1:10 to 1:1000 dilutions, incubated at 37° C. for 10 minutes,and quenched by snap freezing. A "G" map, using RNase T1, and baseladder, using 50 mM Na₂ CO₃ buffer (pH 9), were prepared. The digestionproducts were resolved by sequencing polyacrylamide gel electrophoresis(PAGE). This method provided information regarding the preferredhybridization sites for oligonucleotides on the target ras RNA.Preferred hybridization sites are shown in Table II.

                  TABLE II                                                        ______________________________________                                        Target sites identified by                                                    RNase H affinity mapping                                                      Target Site    SEQ ID NO: Sequence                                            ______________________________________                                        1              9          UGGUGGGCGC                                          2              10         GGCAAGAGUG                                          3              11         CGUCGGUGUG                                          4              12         GUCGGUGUGG                                          ______________________________________                                    

EXAMPLE 18

RNase H Affinity Mapping of HCV RNA by RNAse H Cleavage Mapping

The 5'-untranslated region (UTR) of Hepatitis C virus (HCV) wasenzymatically synthesized as two transcript fragments of 370 and 200nucleotides. These transcripts overlap by 130 nucleotides, and togetherencompass the entire 5'-UTR of HCV. The two transcripts were ³² Pend-labeled according to Sambrook et al., Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2,pg. 11.31-11.32, and gel-purified. Each transcript comprising the 5'-UTRwas incubated at a concentration of approximately 1 μM to 10 nM withrandom oligonucleotide pools synthesized in accordance with the methoddescribed in Example 1 at individual oligonucleotide concentrations of100 pM in 10 mM Tris buffer (pH 8) containing 50 mM NaCl and 5 mM MgCl₂.The hybridization was carried out for at least 16 hours at 37° C. RNaseH (1 U/μL) was added in 1:10 to 1:1000 dilutions, incubated at 37° C.for 10 minutes, and quenched by snap freezing. A "G" map, using RNaseT1, and base ladder, using 50 mM Na₂ CO₃ buffer (pH 9), were prepared.The digestion products were resolved by sequencing polyacrylamide gelelectrophoresis (PAGE) in order to identify hybridization sites ofoligonucleotides on each of the HCV RNA transcripts. The preferredhybridization site information was then used to synthesize complementaryoligonucleotides which bind to this site in order to optimize theirbinding to the target RNA.

EXAMPLE 19

RNase ONE Footprinting Assay with HCV RNA

Two transcript fragments (370 and 200 nucleotides in length, whichoverlap by 200 nucleotides) that encompass the entire 5'-untranslatedregion (5'-UTR) of Hepatitis C virus (HCV) were incubated at aconcentration of 3-30 pM with oligonucleotides that were complementaryto the preferred hybridization sites on the target. Theseoligonucleotides were synthesized in accordance with the methoddescribed in Example 1 at a concentration of 10 μM in 10 mM Tris buffer(pH 8) containing So mM NaCl and 5 mM MgCl₂. The hybridization wascarried out for at least 16 hours at 37° C. RNase ONE (10 U/μL, Promega)was added in 1:2000 to 1:100,000 dilutions, incubated at 25° C. for 5minutes, and quenched by snap freezing. A "G" map, using RNase T1, andbase ladder, using 50 mM Na₂ CO₃ buffer (pH 9), were prepared. Thedigestion products were resolved by sequencing PAGE in order to identifyat least one oligonucleotide that exhibits an RNase ONE footprint at 10μM. The K_(d) for an oligonucleotide of interest was then determined bytitrating that oligonucleotide, at concentrations ranging from 100 pM to10 μM, with RNase ONE. The digestion products were separated bysequencing PAGE and the percent protection afforded by theoligonucleotide was plotted as a function of the oligonucleotideconcentration. The concentration of oligonucleotide at which 50%protection is observed is the K_(d) for that oligonucleotide ofinterest. Using this method, oligonucleotides with optimal binding andspecificity for the target HCV RNA were identified. The K_(d) values,base sequences and sequence ID numbers for these oligonucleotides areshown in Table III.

                  TABLE III                                                       ______________________________________                                        Oligonucleotides having preferred hybridization                               sites on HCV RNA                                                              SEQUENCE       K.sub.d (M)                                                                            SEQ ID NO:                                            ______________________________________                                        GAT CTA TGG T  1 × 10.sup.-8                                                                    13                                                    GTG ATC TAT G  5 × 10.sup.-7                                                                    14                                                    ______________________________________                                    

EXAMPLE 20

RNase ONE Footprinting Assay with ras RNA

The ras 47-mer stem/loop RNA was incubated at a concentration of 3-30 pMwith oligonucleotides that were complementary to the preferredhybridization sites on the target. These oligonucleotides weresynthesized in accordance with the method described in Examples 1 and 2,or with commercially available (Glen Research) 2'-O-methyl amidites, ata concentration of 10 μM in 10 mM Tris buffer (pH 8) consisting of 50 mMNaCl and 5 MM MgCl₂. The hybridization was carried out for at least 16hours at 37° C. RNase ONE (10 U/μL, Promega) was added in 1:2000 to1:100,000 dilutions, incubated at 25° C. for 5 minutes, and quenched bysnap freezing. A "G" map, using RNase T1, and base ladder, using 50 mMNa₂ CO₃ buffer (pH 9), were prepared. The digestion products wereresolved by sequencing PAGE in order to identify at least oneoligonucleotide that exhibits an RNase ONE footprint at 10 μM. The K_(d)for an oligonucleotide of interest was then determined by titrating thatoligonucleotide, at concentrations ranging from 100 pM to 10 μM, withRNase ONE. The digestion products were separated by sequencing PAGE andthe percent protection afforded by the oligonucleotide was plotted as afunction of the oligonucleotide concentration. The concentration ofoligonucleotide at which 50% protection is observed is the K_(d) forthat oligonucleotide of interest. Using this method, oligonucleotideswith enhanced affinity and specificity for the target ras RNA wereidentified. The K_(d) values, base sequences and sequence ID numbers forthese oligonucleotides are shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        Oligonucleotides having preferred                                             hybridization sites on ras RNA                                                ISIS # SEQUENCE     K.sub.d (M)  SEQ ID NO:                                   ______________________________________                                        3271   GCG CCC ACC A                                                                              1 × 10.sup.-6                                                                        15                                           3284   CAC UCU UGC C                                                                              0.33 × 10.sup.-5                                                                     16                                           3291   CAC ACC GAC G                                                                              0.33 × 10.sup.-10                                                                    17                                           4272   CCA CAC CGA C                                                                              0.33 × 10.sup.-10                                                                    18                                           ______________________________________                                    

EXAMPLE 21

Affinity Mapping of HIV RNA by RNase H Cleavage Mapping

The HIV RNA transcripts (rev, tar and 5' long terminal repeat regions)were enzymatically synthesized, ³² P end-labeled according to Sambrooket al., Molecular Cloning. A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989, Volume 2, pg. 11.31-11.32, and gel-purified. TheHIV target was incubated at a concentration of approximately 1 μM to 10nM with random oligonucleotide pools synthesized in accordance with themethod described in Example 1 at individual oligonucleotideconcentrations of 100 pM in 10 mM Tris buffer (pH 8) containing 50 mMNaCl and 5 mM MgCl₂. The hybridization was carried out for at least 16hours at 37° C. RNase H (1 U/μL) was added in 1:10 to 1:1000 dilutions,incubated at 37° C. for 10 minutes, and quenched by snap freezing. A "G"map, using RNase T1, and base ladder, using 50 mM Na₂ CO₃ buffer (pH 9),were prepared. The digestion products were resolved by sequencingpolyacrylamide gel electrophoresis (PAGE). This method providedinformation regarding the preferred hybridization sites foroligonucleotides on the target HIV RNA, and are shown in Table VI.

                  TABLE VI                                                        ______________________________________                                        Target sites identified on HIV RNA                                            by RNase H affinity mapping                                                   Target Site           Sequence                                                ______________________________________                                        1                     GAAG                                                    2                     AGGA                                                    3                     GCAG                                                    4                     GGCA                                                    5                     GAGAG                                                   6                     GCCCG                                                   ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 18                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #  18              TT                                                         - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #             27   TTTT TTTTTTU                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                - 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What is claimed is:
 1. A method of specifically detecting a chemical ordrug in a sample comprising contacting the sample with anoligonucleotide having specific activity for a target biomolecule,wherein said oligonucleotide is identified by the following steps:(a)preparing a set of randomized oligonucleotides; (b) subfractionating theset of randomized oligonucleotides to provide subfractions of saidoligonucleotides; (c) assaying the subfractions of randomizedoligonucleotides for activity against a target biomolecule; (d)selecting the subfraction having the highest activity; (e) assaying theoligonucleotides of the selected subfraction for activity against atarget biomolecule; (f) separating active from inactiveoligonucleotides; (g) recovering said active oligonucleotides; (h)extending a polyA on said active oligonucleotide in a 3' direction toform a first strand; (i) hybridizing said first strand to a firstchimeric primer having a 5' known sequence and a 3' polydT portion; (j)forming a cDNA strand, complementary to the active oligonucleotide,using the first chimeric primer; (k) extending said cDNA formed in (j)in a 3' direction by the addition of polyA; (l) separating saidoligonucleotide and cDNA strand; (m) hybridizing the polyA portion ofsaid cDNA strand to a second chimeric primer having a 5' known sequenceand a 3' polydT portion; (n) filling in the recessed 3' ends to form twocomplementary strands; (o) separating the strands; (p) amplifying thestrands formed in (m) using the first and second chimeric primers bypolymerase chain reaction; (q) excising active oligonuclcotide fromflanking regions; (r) recovering active oligonucleotide; and (s)performing steps (b) through (r) iteratively to determine an activeoligonucleotide; anddetecting the presence or absence of binding,whereby the presence of binding is indicative of the presence of thechemical or drug in said sample.
 2. A method of specifically detecting achemical or drug in a sample comprising contacting the sample with anoligonucleotide having specific activity for a target biomolecule,wherein said oligonucleotide is identified by the following steps:(a)preparing a set of randomized oligonucleotides; (b) assaying the set ofrandomized oligonucleotides for activity against a target biomolecule;(c) separating active from inactive oligonucleotides; (d) recoveringactive oligonucleotide; (e) extending a polyA on said activeoligonucleotide in a 3' direction to form a first strand; (f)hybridizing said first strand to a first chimeric primer having a 5'known sequence and a 3' polydT portion; (g) forming a cDNA strandcomplementary to active oligonuclcotide, using the first chimericprimer; (h) extending said cDNA formed in (g) in a 3' direction by theaddition of polyA; (i) separating said oligonucleotide and cDNA strand;(j) hybridizing the polyA potion of said cDNA strand to a secondchimeric primer having a 5' known sequence and a 3' polydT portion; (k)filling in the recessed 3' ends to form two complementary strands; (l)separating the strands; (m) amplifying the strands formed in (k) usingthe first and second chimeric primers by polymerase chain reaction; (n)excising active oligonucleotide; (o) recovering active oligonucleotide;and (p) performing steps (b) through (o) iteratively to determine anoligonucleotide having specific activity for a target biomolecule;anddetecting the presence or absence of binding, whereby the presence ofbinding is indicative of the presence of the chemical or drug in saidsample.
 3. A method of specifically detecting a chemical or drug in asample comprising contacting the sample with an oligonucleotide havingspecific activity for a target biomolecule, wherein said oligonucleotideis identified by the following steps:(a) preparing a set of randomizedoligonucleotides; (b) subfractionating the set of randomizedoligonucleotides to provide subfractions of said oligonucleotides; (c)assaying each of the subfractions of randomized oligonucleotides foractivity against a target biomolecule; (d) selecting the subfractionhaving the highest activity; (e) assaying the oligonucleotides of theselected subfraction for activity against a target biomolecule; (f)separating active from inactive oligonucleotides; (g) recovering saidactive oligonucleotides; (h) amplifying the active oligonucleotidesrecovered in (d); and (i) determining the nucleic acid sequence of saidamplified oligonucleotides; anddetecting the presence or absence ofbinding, whereby the presence of binding is indicative of the presenceof the chemical or drug in said sample.
 4. A method of specificallydetecting a chemical or drug in a sample comprising contacting thesample with an oligonucleotide having specific activity for a targetbiomolecule, wherein said oligonucleotide is identified by the followingsteps:(a) preparing a set of randomized oligonucleotides; (b) assayingthe set of randomized oligonucleotides for activity against a targetbiomolecule; (c) separating active from inactive oligonucleotides; (d)recovering said active oligonucleotides; (e) amplifying the activeoligonucleotides; and (f) determining the nucleic acid sequence of saidamplified oligonucleotides; anddetecting the presence or absence ofbinding, whereby the presence of binding is indicative of the presenceof the chemical or drug in said sample.
 5. A method of specificallydetecting a chemical or drug in a sample comprising contacting thesample with an oligonucleotide having specific activity for a targetbiomolecule, wherein said oligonucleotide is identified by the followingsteps:(a) preparing a set of randomized oligonucleotides; (b) assayingthe set of randomized oligonucleotides for activity against a targetbiomolecule; (c) separating active from inactive oligonucleotides; (d)recovering said active oligonucleotides; (e) characterizing therecovered oligonucleotides to provide an oligonucleotide cassette; (d)preparing a set of oligonucleotides comprising a cassette and at leastone flanking region of randomized positions; (g) assaying the set ofoligonucleotides for activity against a target biomolecule; (h)separating active from inactive oligonucleotides; (i) recovering saidactive oligonucleotides; (f) characterizing the recoveredoligonucleotides to provide an oligonucleotide cassette; and (k)performing steps (f) through (j) iteratively to determine anoligonucleotide having specific activity for a target biomolecule;anddetecting the presence or absence of binding, whereby the presence ofbinding is indicative of the presence of the chemical or drug in saidsample.
 6. A method of specifically detecting a chemical or drug in asample comprising contacting the sample with an oligonucleotide havingspecific activity for a target biomolecule, wherein said oligonuclcotideis identified by the following steps:(a) preparing a set of randomizedoligonucleotides; (b) subfractionating the set of randomizedoligonucleotides to provide subfractions of said oligonucleotides; (c)assaying each of the subfractions of randomized oligonucleotides foractivity against a target biomolecule; (d) selecting the subfractionhaving the highest activity; (e) assaying the oligonucleotides of theselected subfraction for activity against a target biomolecule; (f)separating active from inactive oligonucleotides; (g) recovering saidactive oligonucleotides; and (h) characterizing the recoveredoligonucleotides by microanalytical structural determination;anddetecting the presence or absence of binding, whereby the presence ofbinding is indicative of the presence of the chemical or drug in saidsample.
 7. A method of specifically detecting a chemical or drug in asample comprising contacting the sample with an oligonucleotide havingspecific activity for a target biomolecule, wherein said oligonucleotideis identified by the following steps:(a) preparing a set of randomizedoligonucleotides; assaying the set of randomized oligonucleotides foractivity against a target biomolecule; (c) separating active frominactive oligonucleotides; (d) recovering said active oligonucleotides;and (e) characterizing the recovered oligonucleotides by microanalyticalstructural determination; anddetecting the presence or absence ofbinding, whereby the presence of binding is indicative of the presenceof the chemical or drug in said sample.